A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

Discrete-time semi-Markov modeling of human papillomavirus persistence.

Multi-state modeling is often employed to describe the progression of a disease process. In epidemiological studies of certain diseases, the disease state is typically only observed at periodic clinical visits, producing incomplete longitudinal data. In this paper we consider fitting semi-Markov models to estimate the persistence of human papillomavirus (HPV) type-specific infection in studies where the status of HPV type(s) is assessed periodically. Simulation study results are presented indicating that the semi-Markov estimator is more accurate than an estimator currently used in the HPV literature. The methods are illustrated using data from the HIV Epidemiology Research Study.

Breast radiotherapy: considerations in older patients.

With an ageing population, the number of older women with breast cancer eligible for adjuvant irradiation after breast conserving surgery and mastectomy is rising. There is a dearth of level 1 data on the effect of adjuvant irradiation on local control, quality of life and survival. In large part this reflects the exclusion of patients over the age of 70 years from randomised trials. The prevention of local recurrence may reduce the risks of dissemination. However, older women with early breast cancer and a life expectancy of less than 5 years are unlikely to derive a survival benefit from adjuvant radiotherapy. Rates of access of older patients to adjuvant irradiation are lower than for younger patients. Physician and patient bias and co-morbidities are contributory factors. There are also competing risks of mortality from co-morbidities, particularly in women over the age of 80 years. Postoperative radiotherapy after breast conserving surgery does not seem to compromise overall quality of life of older patients. Although the absolute reduction in local recurrence from adjuvant radiotherapy is modest in lower risk older patients after breast conserving surgery and adjuvant systemic therapy, there has to date been no group of fitter old patients defined from whom radiotherapy can be reasonably omitted. Guidelines for postmastectomy radiotherapy should not differ from younger patients. Adequately powered randomised trials are needed to assess the effect of adjuvant irradiation in older patients on outcomes after breast conserving surgery and mastectomy to provide a more robust basis for evidence-based radiotherapy practice.

Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice.

BACKGROUND: The mechanisms responsible for thrombocytopenia associated with dengue fever (DF) and dengue hemorrhage fever (DHF) remain unclear. OBJECTIVE: In this study, we investigated the pathogenic effects of dengue virus (DENV) non-structural protein 1 (NS1) on the elicitation of platelet cross-reactive antibodies. RESULTS: The results showed that anti-DENV NS1 immunoglobulins (Igs) derived from both patients with DF/DHF and recombinant NS1-immunized rabbits could opsonize normal human platelets and enhance platelet-macrophage engagements in vitro. In addition, treatments with anti-NS1 Igs abnormally activated human platelets and induced thrombocytopenia in mice. These prothrombotic characteristics of anti-NS1 Ig might increase the disease burden of coagulant-aberrant DHF patients. To test this hypothesis, we injected anti-NS1 Igs into C57BL/6J mice that were preconditioned into a hypercoagulable state by warfarin treatments. When given before but not after platelet-lysate pre-adsorption, the anti-NS1 Igs injection treatments significantly increased mortality, fibrin deposition in lung, and plasma D-dimer levels, but significantly decreased anticoagulant proteins C, protein S and antithrombin III. CONCLUSIONS: These results suggest that the platelet-bound antibody fractions of anti-NS1 Ig are prothrombotic, which might exacerbate the severity of disease in hosts with an imbalanced coagulant system.

Assessment of intervention measures for the 2003 SARS epidemic in Taiwan by use of a back-projection method.

OBJECTIVES: To reconstruct the infection curve for the 2003 severe acute respiratory syndrome (SARS) epidemic in Taiwan and to ascertain the temporal changes in the daily number of infections that occurred during the course of the outbreak. METHOD: Back-projection method. RESULTS: The peaks of the epidemic correspond well with the occurrence of major infection clusters in the hospitals. The overall downward trend of the infection curve after early May corresponds well to the date (May 10) when changes in the review and classification procedure were implemented by the SARS Prevention and Extrication Committee. CONCLUSION: The major infection control measures taken by the Taiwanese government over the course of the SARS epidemic, particularly those regarding infection control in hospitals, played a crucial role in containing the outbreak.

A randomised controlled trial of postoperative radiotherapy following breast-conserving surgery in a minimum-risk older population. The PRIME trial.

OBJECTIVES: To assess whether omission of postoperative radiotherapy in women with 'low-risk' axillary node negative breast cancer (T0-2) treated by breast-conserving surgery and endocrine therapy improves quality of life and is more cost-effective. DESIGN: A randomised controlled clinical trial, using a method of minimisation balanced by centre, grade of cancer, age, lymphatic/vascular invasion and preoperative endocrine therapy, was performed. A non-randomised cohort was also recruited, in order to complete a comprehensive cohort study. SETTING: The setting was breast cancer clinics in cancer centres in the UK. PARTICIPANTS: Patients aged 65 years or more were eligible provided that their cancers were considered to be at low risk of local recurrence, were suitable for breast-conservation surgery, were receiving endocrine therapy and were able and willing to give informed consent. INTERVENTIONS: The standard treatment of postoperative breast irradiation or the omission of radiotherapy. MAIN OUTCOME MEASURES: Quality of life was the primary outcome measure, together with anxiety and depression and cost-effectiveness. Secondary outcome measures were recurrence rates, functional status, treatment-related morbidity and cosmesis. The principal method of data collection was by questionnaire, completed at home with a research nurse at four times over 15 months. RESULTS: The hypothesised improvement in overall quality of life with the omission of radiotherapy was not seen in the EuroQol assessment or in the functionality and symptoms summary domains of the European Organisation for Research in the Treatment of Cancer (EORTC) scales. Some differences were apparent within subscales of the EORTC questionnaires, and insights into the impact of treatment were also provided by the qualitative data obtained by open-ended questions. Differences were most apparent shortly after the time of completion of radiotherapy. Radiotherapy was then associated with increased breast symptoms and with greater fatigue but with less insomnia and endocrine side-effects. Patients had significant concerns about the delivery of radiotherapy services, such as transport, accommodation and travel costs associated with receiving radiotherapy. By the end of follow-up, patients receiving radiotherapy were expressing less anxiety about recurrence than those who had not received radiotherapy. Functionality was not greatly affected by treatment. Within the randomised controlled trial, the Barthel Index demonstrated a small but significant fall in functionality with radiotherapy compared with the no radiotherapy arm of the trial. Results from the non-randomised patients did not confirm this effect, however. Cosmetic results were better in those not receiving radiotherapy but this did not appear to be an important issue to the patients. The use of home-based assessments by a research nurse proved to be an effective way of obtaining high-quality data. Costs to the NHS associated with postoperative radiotherapy were calculated to be of the order of 2000 pounds per patient. In the follow-up in this study, there were no recurrences, and the quality of life utilities from EuroQol were almost identical. CONCLUSIONS: Although there are no differences in overall quality of life scores between the patients treated with and without radiotherapy, there are several dimensions that exhibit significant advantage to the omission of irradiation. Over the first 15 months, radiotherapy for this population is not a cost-effective treatment. However, the early postoperative outcome does not give a complete answer and the eventual cost-effectiveness will only become clear after long-term follow-up. Extrapolations from these data suggest that radiotherapy may not be a cost-effective treatment unless it results in a recurrence rate that is at least 5% lower in absolute terms than those treated without radiotherapy. Further research is needed into a number of areas including the long-term aspects of quality of life, clinical outcomes, costs and consequences of omitting radiotherapy.

When may adjuvant radiotherapy be avoided in operable breast cancer?

Randomised trials in which the omission of radiotherapy has been tested after breast-conserving surgery, with or without adjuvant systemic therapy, show a significant four- to five-fold reduction in local recurrence. As yet, no subgroup of women managed by breast-conserving surgery has been identified from whom radiotherapy can be withheld. Few randomised data have been published on the effect of omission of radiotherapy on local control, quality of life and costs, particularly in older women for whom the risk of local recurrence is generally lower. Ongoing trials are evaluating the role of radiotherapy in this population of low risk, older women. Adjuvant radiotherapy after breast-conserving surgery or mastectomy significantly reduces the incidence of local recurrence. In women who have had a mastectomy at high risk of recurrence (> 20% risk of recurrence at 10 years), adjuvant radiotherapy improves survival if combined with adjuvant systemic therapy. Among women with T3 tumours, and those with four or more involved axillary nodes treated by mastectomy, postoperative radiotherapy is the standard of care. For women at intermediate risk of recurrence (i.e. <15% 10-year risk of recurrence after surgery and systemic therapy alone), with one to three involved nodes or node negative with other risk factors, the role of radiotherapy is unclear. Clinical trials to assess the role of postmastectomy radiotherapy (PMRT) in this setting are needed. For pT1-2, pNO tumours without other risk factors, there is no evidence at present that PMRT is needed.

Fractal and chaotic dynamics in nervous systems.

The chaotic aspects of brain structure and dynamics have been discussed. The relation of chaos to fractal processes in the brain from the neurosystems level down to the molecule has been explored. It is found that chaos appears to play an integral, though not necessarily exclusive role in function at all levels of organization from the neurosystems to the molecular and quantum levels. An interesting consequence involving the possible interface between chaotic dynamics and quantum physics has been discussed because of its potential significance is resolving several of the most intractable conceptual problems to do with computability, the brain and the mind (Blakemore and Greenfield, 1987; Hooper and Teresi, 1987; Rose, 1973; Searle, 1979; Penrose, 1986, 1989).

Secretion and inactivation of myeloperoxidase by isolated neutrophils.

Neutrophils prevent infection by ingesting and killing microorganisms but oxidants and proteases released by neutrophils damage host tissues. Our aim was to identify factors that regulate oxidant production by the enzyme myeloperoxidase (MPO) following secretion of MPO into the medium. Cells stimulated with phorbol myristate acetate (PMA) or opsonized zymosan particles secreted MPO and released superoxide free radicals (.O2-). Dismutation of .O2- produced hydrogen peroxide (H2O2) and MPO catalyzed the oxidation of chloride ion by H2O2 to produce the toxic oxidant hypochlorous acid (HOCl). Adding the enzyme superoxide dismutase (SOD) to increase the rate of conversion of .O2- to H2O2 had pH-dependent effects on HOCl production. From pH 6.0 to 7.4, SOD promoted HOCl production by up to 500% but SOD had no effect at pH 7.6 and inhibited by 40 +/- 10% at pH 7.8. In further experiments at pH 7.0, MPO activity in the cells decreased by 25 +/- 2 and 44 +/- 4% during 1-h incubations with PMA and zymosan. Only 1 +/- 0 and 3 +/- 1% of the total activity was found in the medium, indicating that most of the secreted MPO was inactivated. Loss of activity was not accompanied by proteolytic destruction of the MPO protein, which was measured with anti-MPO antibodies. SOD raised the amount of active MPO in the medium two- to sevenfold, but adding deferoxamine to chelate iron or adding ferric ion had no effect. The ionophore A23187 was as effective as zymosan as a stimulus for MPO secretion but .O2- production by ionophore-stimulated cells was less than 4% of that of PMA- or zymosan-stimulated cells and most of the secreted MPO was found active in the medium. When PMA-stimulated cells were incubated with purified MPO, the added MPO activity was lost from the medium. Binding or proteolysis did not account for loss of activity as indicated by recovery of added radioiodinated MPO from the medium. The visible absorption spectrum of MPO was lost, indicating destruction of the iron-containing prosthetic group. Loss of activity and loss of the MPO spectrum were blocked by SOD but not by deferoxamine or catalase. The results indicate that, in the physiological pH range, inactivation of MPO in the medium suppressed HOCl production. Inactivation required O2- but not HOCl, H2O2, or free iron. Inactivation of secreted MPO may limit MPO-mediated damage to host tissues by stimulated neutrophils.

Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

Community-wide epidemiological investigation of a typhoid outbreak in a rural township in Taiwan, Republic of China.

An outbreak of typhoid fever occurred in Chu-Tung township, Taiwan, with dates of onset from 6 July to 8 August, 1983. Fifty-four cases were hospitalized, of which 52 were laboratory confirmed. A chloramphenicol-resistant strain of Salmonella typhi was isolated from patients' blood samples. A community survey of 2772 people selected from 490 households by stratified systematic cluster sampling, presented an attack rate of 9.4 per 1000 and a case reporting rate of 10%. The attack rate was higher in males than females for persons younger than ten years, but was greater in females than in males aged 40 years and older. The only consistent characteristic of the early outbreak cases was drinking of tapwater (10/10, 100% versus 319/490, 65% of the controls). None of the early cases but 36% (13/36) of the late cases had drunk stream or river water. Households of early cases had better hygienic conditions than those of late cases. Laboratory examination of environmental specimens indicated Escherichia coli contamination of tapwater, well water and all stream foci associated with human activities. The epidemiological data combined with laboratory results suggested that the epidemic might be due to repeated contamination of some common source (such as municipal tapwater) and/or a variety of other vehicles.

Seroepidemiology and evaluation of passive surveillance during 1988-1989 measles outbreak in Taiwan.

A seroepidemiological study was carried out to explore the risk factors of a measles outbreak that occurred among school children at a rural village (Li-Tse) in Taiwan. Among the 1166 participants, the percentage susceptible before the outbreak was 10.5% (122/1158) which was estimated as the sum of measles IgG-negative (29/1158) and IgM-positive (93/1166) individuals. Among 340 vaccinated children, 16 (4.7%) were measles IgM-positive and 10 (2.9%) were measles IgG-negative; therefore the vaccine failure rate was estimated to be 7.6% (26/340) and vaccine efficacy was 79.7% (95% confidence interval [CI] : 65.0-88.5). The most important risk factors for acquiring measles infection were the presence of other measles cases in the family (Odds Ratio [OR] = 32.5, P = 0.002) and the presence of more than two cases in a class (OR = 29.1, P = 0.003). The physician reporting rate was 6.1% (4/66), and the sensitivity of passive measles surveillance was only 4.3% (4/93) by active serosurvey. A concomitant rubella epidemic also amplified the inaccuracy of a passive reporting system based only on clinical diagnosis. Five children developed measles IgM but did not experience any symptoms, indicating that asymptomatic measles infection can occur. Our experience has highlighted three important areas for future measles elimination: (1) the need for serological evaluation of vaccinees, particularly those who were born during the introduction of mass immunization; (2) improvement in measles vaccine efficacy; and (3) further investigations on the role of asymptomatic transmission and susceptibles who remain after mass immunization.

ORP3 splice variants and their expression in human tissues and hematopoietic cells.

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Silent transmission of the dengue virus in southern Taiwan.

Immunoglobulin M (IgM) antibody to dengue virus was examined from a total of 3,099 serum samples collected in southern Taiwan. Of 1,232 sera collected from a junior high school and four elementary schools in Liu-Chiu, 35 were IgM-positive, demonstrating that the dengue virus has been circulating on the island, despite the fact that no epidemic has been reported in the past 10 years. Sixteen of 925 sera collected from three elementary schools in Tung-Kang in 1991 were found to be IgM-positive and two of 192 sera from adults in the local community were positive. The IgM-positive subjects tended to be aggregated around a port. Fishing boats that had stopped in neighboring endemic countries were presumed to have introduced the virus periodically, causing a low level of inapparent infections. In the Kaohsiung area, two of 108 suspected clinical cases and four of 642 community-based sera were IgM-positive. Rapid urbanization has provided appropriate circumstances for vector breeding in this area and the high population density has also increased contact frequency between humans and mosquito vectors. This has, in turn, increased the possibility of silent transmission of the dengue virus via either intermittent reintroduction of the virus or continuation of inapparent infections or both. Establishment of a early warning system using the IgM antibody capture-enzyme-linked immunosorbent assay is suggested for effective monitoring of the disease.

High-dose cisplatin with diethyldithiocarbamate (DDTC) rescue therapy: preliminary pharmacologic observations.

Diethyldithiocarbamate (DDTC), a chelating agent that is a major metabolite of disulfuram, has been proposed as a potential rescue agent to reduce toxicity following high-dose cisplatin (HDCP) therapy. In the present study, we examined the pharmacologic interaction of HDCP and DDTC given as rescue therapy. Total plasma platinum and ultrafiltrate platinum pharmacokinetics and DDTC levels were determined in six patients with advanced malignancies who received a total of 11 cycles of HDCP with DDTC rescue. HDCP therapy (200 mg/m2 per cycle) consisted of 100 mg/m2 reconstituted in 250 cc 3% saline and infused over 3 h on days 1 and 8 of each 28-day cycle. DDTC rescue at a dose of 4 gm/m2 was given by an i.v. infusion (duration 1.5-3.5 h), beginning 45 min after the completion of cisplatin infusion. Peak total and ultrafiltrate levels and cisplatin pharmacokinetics in this study were indistinguishable from those of previous studies using the same HDCP regimen without DDTC rescue. Ultrafiltrate or unbound plasma platinum was less than 10% of total plasma platinum concentrations and demonstrated a biphasic pattern of elimination. Levels of DDTC predicted to be chemoprotective (greater than 400 microM) were achieved with the dose and schedule used in this study. These data demonstrate that DDTC can be targeted to protective plasma concentrations without significantly altering plasma cisplatin pharmacokinetics and support the potential usefulness of DDTC as a rescue agent following HDCP therapy.

Leukocyte myeloperoxidase and salivary lactoperoxidase: identification and quantitation in human mixed saliva.

Human salivary lactoperoxidase (HS-LP) is synthesized and secreted by the salivary glands, whereas myeloperoxidase (MPO) is found in PMN leukocytes, which migrate into the oral cavity at gingival crevices. HS-LP levels vary with changes in salivary gland function, but increased numbers of MPO-containing leukocytes indicate infection or inflammation of oral tissues. To determine the contribution of each enzyme to the peroxidase activity of mixed-saliva samples, activity was assayed at pH 5.4 with tetramethylbenzidine as the substrate, with and without the inhibitor dapsone (4,4'-diaminodiphenylsulfone). Dapsone blocked the activity of HS-LP but not MPO. The enzymes were also separated and partially purified from the soluble portion of saliva samples and from detergent extracts of the saliva sediment. Chromatographic properties of the proteins were similar to those of LP from bovine milk (BM-LP) and MPO from human leukocytes. The identity and amounts of the enzymes were confirmed by the absorption spectra and by immunoblotting with antibodies to BM-LP and human MPO. Eosinophil peroxidase (EPO), a distinct enzyme found in eosinophilic leukocytes, was not detected by chromatography or with antibodies to human EPO. On average, 75% of the activity in samples from normal donors was due to MPO and 25% to HS-LP. When corrected for the lower specific activity of HS-LP in this assay, the average amount of MPO (3.6 micrograms/mL) was twice the amount of HS-LP (1.9 micrograms/mL). The amount of MPO corresponded to 1 x 10(6) PMN leukocytes/mL of saliva. The enzymes were distributed differently: Eighty-nine percent of the HS-LP was in the soluble portion of saliva, and 78% of the MPO was in the sediment, which contained 51% of the total activity. In contrast to results obtained with PMN leukocytes from blood, detergent was not required for MPO activity to be measured in saliva, indicating that the enzyme was accessible to peroxidase substrates. The results indicate that MPO is responsible for a large portion of peroxidase-catalyzed reactions in mixed saliva. The unique function of HS-LP may be carried out within the salivary glands, prior to secretion into the oral cavity.

What is the evidence for a reduced risk of local recurrence with age among older patients treated by breast conserving therapy?

With the rising age of the population and the proposed extension of the breast screening programme to older women, increasing numbers of older patients are becoming eligible for breast conserving surgery and post-operative breast irradiation. Women over the age of 70 have traditionally been omitted from randomized controlled trials for assessing the role of breast radiotherapy after local surgery. The majority of trials suggest that local recurrence rates do decline with age. Similar conclusions are suggested by many non-randomized studies. Comparison of randomized and non-randomized studies is limited by differing extent of classifying tumour margins, nodal status, use of adjuvant systemic therapy, sample size, analytical approaches and duration of follow-up. Large randomized trials in older women are needed to assess whether, with careful attention to obtaining clear tumour margins, radiotherapy is required in low risk, ER positive, node negative breast cancer patients following wide excision and adjuvant tamoxifen. Within both randomized and non-randomized studies, only a few studies have failed to demonstrate an impact of age on recurrence rates following breast conserving treatment, with the majority finding a reduction in local recurrence rates with increasing age. Importantly for interpretation, no studies suggest that recurrence rates increase with age. The variation in analytical approaches and sample sizes are such that the variety of conclusions is not surprising. The results are compatible with a tendency for local recurrence rates to fall with age, but the variability is such that one cannot quantify this change with any precision.

Myeloperoxidase-catalyzed chlorination of histamine by stimulated neutrophils.

To investigate neutrophil interactions with mediators released by mast cells at sites of inflammation, stimulated neutrophils were incubated with histamine. No accumulation of chlorinated histamine derivatives was detected in the medium. Instead, histamine inhibited the formation of chloramine derivatives of other amines. Incubation with radiolabeled histamine resulted in rapid uptake of label into the cells, and most of the label could be extracted and recovered as histamine. About 3% of the label taken up was incorporated into acid-precipitable forms. Uptake depended on myeloperoxidase (MPO)-catalyzed formation of chlorinating agents. Uptake was promoted by adding MPO and blocked by the MPO inhibitor dapsone, catalase, scavengers for hypochlorous acid and chloramines, or in a low-chloride medium, but not by histamine receptor antagonists. Incubation of histamine with MPO, hydrogen peroxide, and chloride resulted in formation of mono- and dichloramine derivatives of the primary amino group. Above pH 7.0, the chloramines were primarily in uncharged, lipophilic forms as indicated by partitioning into organic solvents. Histamine is a cation at neutral pH, but chlorination eliminated the charge on the amino group and shifted the pKa of the imidazole ring, resulting in formation of neutral histamine-chloramines. Incubation of neutrophils or other blood cells with radiolabeled histamine-chloramines resulted in rapid uptake of label, indicating membrane permeation by the uncharged, lipid-soluble forms. Incubation with labeled histamine-dichloramine also resulted in acid-precipitable incorporation. The results indicate that MPO-catalyzed chlorination of histamine could modulate histamine activity, tissue distribution, and metabolism at sites of inflammation.

Amplification of viral RNA for the detection of dengue types 1 and 2 virus.

In vitro DNA amplification by means of the polymerase chain reaction (PCR) was used to amplify dengue types 1 and 2 viral genomes in cultured cells and in the serum of persons infected with dengue virus. Results of the present investigation suggest that the PCR method is type-specific in detecting dengue virus and has a detection sensitivity of less than 100 plaque-forming units (pfu) for both serotypes of the virus. The PCR method may be useful for detecting and typing dengue virus in clinical and epidemiological specimens.